Ovarian folliculogenesis is a dynamic process marked by exponential expansion and differentiation of the granulosa cells, maturation of the oocyte, and neovascularization. Although the central roles of gonadotropins and of gonadal steroids in this explosive agenda are well accepted, the variable fate of follicles within the same ovary suggests the existence of additional intraovarian modulatory systems.¹ Stated differently, it is presumed that gonadotropin action is “fine tuned” in situ, thereby accounting for observed differences in the rate and extent of development of ovarian follicles. Alterations in gonadotropin secretion cannot adequately explain the initiation and arrest of meiosis within the oocyte, the acquisition of follicular dominance, or the failure of follicular development, which leads to atresia. It is likely that the earlier stages of follicular growth, generally considered to be gonadotropin independent, may be controlled by intraovarian signaling.

The concept of local gonadal regulators originated when the embryology of the ovary was the subject of intense scrutiny. Gonadal differentiation was proposed by Witschi to result from the interaction of two morphogenic substances called cortexone and medullarine, the first of which was thought of as the stimulator of ovarian development and the latter as the promoter of testicular growth.² Although multiple other contributors must undoubtedly be acknowledged, the notion of intraovarian regulators was promoted with special vigor by the late Cornelia Post Channing, whose pioneering experiments ushered in contemporary molecular endocrinology as it applies to ovarian physiology.³

Among potential novel intraovarian regulators, growth factors, cytokines, and neuropeptides have been the subject of increasingly intense investigation. Most of these agents are not expected to act in the traditional endocrine fashion because of their local intraovarian generation (as opposed to circulatory-derived influences emanating from distant endocrine glands). Speculation favors the notion that a host of putative intraovarian regulators may engage in subtle in situ modulation and coordination of growth and function of the varied follicular cell types: oocytes, granulosa, theca, and vascular epithelium. In this capacity, a given putative intraovarian regulator may modulate the replication or cytodifferentiation of a developing ovarian cell, acting in its own right or as an amplifier-attenuator of gonadotropin action. Such putative intraovarian regulators may also be concerned with intercompartmental communication, allowing tighter linking of different cellular populations. For example, a growing body of evidence suggests that granulosa cell-derived modulators may regulate the adjacent theca-interstitial cell compartment in the interest of coordinated follicular development. In doing so, the granulosa cell may exert some control over its own destiny, in that it may regulate the very inflow of androgenic substrate from the neighboring theca. Together, gonadotropins, steroids, and locally derived peptidergic principles form a triad, which modulates the growth and differentiation of ovarian follicles (Fig. 1). According to contemporary views, potential intraovarian communication is mostly paracrine or autocrine in nature. Paracrine communication involves local diffusion of regulators from producer cells to distinct target cells within the same organ. This is a heteroregulatory phenomenon that could allow for intercompartmental communication, providing a tighter linkage of different cellular populations. In the ovary, the ability of increasing numbers of granulosa cells to produce estrogen depends on the concomitant ability of the thecal layer to provide the proper amounts of androgenic substrate. The granulosa cell, in the interest of efficient coupling, may elaborate substances (e.g. insulin-like growth factor-I [IGF-I] inhibin, activin) that could alter the function of the neighboring theca.

The other type of cellular communication, autocrine regulation, involves the action of a regulator on surface receptors at its cell of origin. This is a self-regulatory phenomenon wherein a single cell type modulates its own activity. In the ovary, granulosa cells elaborate substances such as IGF-I and activin that can alter granulosa cell function. Whereas steroids may be exerting intracrine (i.e. regulation within the cell of origin) effects, there is no evidence for juxtacrine (i.e. contact-dependent regulation between immediately adjacent cells) effects in the ovary.

To qualify as a bona fide intraovarian regulator, the putative agent needs to meet the minimal criteria of local production, local reception, and local action. Some evidence of indispensability to in vivo ovarian function needs to be provided. For the most part, few of the putative intraovarian regulators under study (Table 1) have satisfactorily met all of the previously described criteria (i.e. IGF-I, activin). Accordingly, the information provided later can be viewed as a prelude to what the future holds. Undoubtedly, additional information will become available with respect to the putative intraovarian regulators under consideration. It is equally certain that novel candidates will be added to this preliminary list, requiring modification of current views.

TABLE 1. Established and Putative Intraovarian Regulators

  Insulin-Like Growth Factor System
  IGF-I
  IGF-II
  IGF binding proteins
  Inhibin/Activin Systems
  Inhibin
  Activin
  Follistatin
  Interleukin-1 System
  Interleukin-1
  Interleukin-1 receptor antagonist
  IL-1 binding protein (IL-1 receptor type II)
  Other Growth Factors
  EGF/TGFα
  TGFβ1, TGFβ2
  NGF
  aFGF, bFGF
  VEGF
  TNFα
  Other Peptidergic Factors
  Ovarian renin angiotensin system
  VIP
  Oxytocin
  Endothelin

The following sections describe a select group of putative intraovarian regulators reflecting different modes of action. The principle action of each regulator is briefly listed in Table 2.

TABLE 2. Principal Actions of Intraovarian Regulators

  Insulin-like growth factor-I

  Follicle-stimulating hormone (FSH) amplification
  Follicular growth
  Follicular selection

  Ovulation induction
  Glycolysis
  Glucose transport

  Inhibits steroidogenesis
  FSH antagonist
  Induces apoptosis/luteolysis
  Ovulation inhibition

A 70-amino acid polypeptide, IGF-I plays a variety of metabolic and endocrine roles, not the least of which is the promotion of linear skeletal growth. In keeping with its ubiquitous distribution, IGF-I is also known to serve a variety of autocrine or paracrine tissue-specific functions to suit the needs dictated by the tissues in question. In this respect, the ovary is but one example of many exemplifying the general concept of intraorgan regulation.⁴

A large body of information now strongly supports the view that the ovary is a site of IGF-I production, reception, and action (Fig. 2). Whereas the rat granulosa cell appears to be the only cellular site of IGF-I gene expression,^5,6 the granulosa^7,8 and the theca-interstitial cells^9,10 possess specific receptors for this peptidergic ligand. The mouse intraovarian IGF-I system is generally comparable to that of the rat, although they differ is several aspects.^11,12 These observations suggest that IGF-I may engage in intercompartmental communication in the interest of coordinated follicular development. IGF-I hormonal action appears subject to further modulation through the local elaboration of low-molecular-weight binding proteins (IGFBPs), the role and regulation of which are receiving increasing attention. The discovery of IGFBP-4 mRNA in early-stage atretic follicles raises the intriguing possibility that depletion of IGF action may be necessary for the onset of the atretic process.¹³ Although multiple ovarian actions have been ascribed to IGF-I, its main role appears to be the amplification of gonadotropin action in thecainterstitial and granulosa cells. All markers of follicle-stimulating hormone (FSH) induction (e.g. production of progesterone inhibin, luteinizing hormone binding) are enhanced by IGF-I. Optimal gonadotropin hormonal action is contingent on the prior availability of granulosa cell-derived IGF-I and the consequent amplification of the gonadotropic signal. Given a hypothetical IGF vacuum created by excess exogenous IGF binding proteins, intrinsic FSH hormonal action proves to be relatively modest (Fig. 3). In contrast, given IGF-replete circumstances, FSH hormonal action in toto may be composed of a modest intrinsic component complemented by a substantial synergistic component.¹⁴

At the clinical level, ovarian IGF-I may have a bearing on the puberty-promoting effect of growth hormone. An association appears to exist between isolated growth hormone deficiency and delayed puberty in rodents and human subjects, a process reversed by systemic hormone replacement therapy. Given that ovarian IGF-I and its receptor may be growth hormone dependent, it is tempting to speculate that the ability of growth hormone to accelerate pubertal maturation in part may be caused by the promotion of ovarian IGF-I production and reception with the consequent local potentiation of gonadotropin action.

Clear evidence for the central role of IGF-I in reproductive physiology has been gained from gene knockout technology. In the mouse, targeted null mutation of the Igf1 gene, encoding IGF-I, results in infertility secondary to failure to ovulate even after administration of gonadotropins.¹⁵ Given the IGF-I primary action of FSH amplification, further efforts have been made to elucidate its mechanism of action in that regard.^16,17,18

Purified on the basis of its ability to stimulate precocious eyelid opening and tooth eruption in newborn mice, epidermal growth factor (EGF) was initially found in male mouse submaxillary glands and later in human urine as urogastrone. Mature EGF comprises a single polypeptide chain of 53 amino acids displaying three internal disulfide bonds. Originally thought to have a limited range of tissue expression, in situ hybridization analysis of sections of whole newborn mice indicate that RNA complementary to cloned EGF probes may be present in a large variety of tissues.

Transforming growth factor-α (TGF-α), a structural analog of EGF, is a single-chain, 50-amino acid polypeptide capable of binding to an apparently common EGF/TGF receptor. EGF and TGF recognize the same cellular receptor, and they are apparently equipotent in most systems studied. EGF may be the adult form of the embryonic growth factor TGF. TGF is a member of a family of polypeptides best known for their ability to produce an acute, albeit reversible, phenotypic transformation of normal mammalian cells. TGF can be defined operationally by its ability to stimulate anchorage-independent growth in soft agar of cells, which are otherwise anchorage dependent.

At the level of the ovary, EGF exerts potent regulatory effects on granulosa cell proliferation and differentiation.^19,20,21 These effects of EGF presumably are mediated by specific cell membrane receptors, the existence of which has been demonstrated on bovine, ovine, and murine granulosa cells.²² However, the identity of the endogenous ligand occupying the receptor in question under in vivo conditions remains uncertain.

TGF, like EGF, proved to be a potent inhibitor of gonadotropin-supported granulosa cell differentiation. TGF has been localized to the thecainterstitial cell compartment,²³ thereby raising the possibility that theca-interstitial cell-derived TGF may exert paracrine effects at the level of the adjacent granulosa cell. Theca-interstitial cell-derived TGF may also engage in autocrine effects.²⁴ It is tempting to speculate that TGF of theca-interstitial cell origin may orchestrate follicular activities at the granulosa and theca-interstitial cell level (Fig. 4). However, because TGF has also been shown to suppress gonadotropin-supported theca-interstitial cell differentiation,²⁴ the possibility of an autocrine mode of action cannot be excluded. Further evidence supports the possibility that TGF may also be expressed by other compartments of the ovary (e.g. granulosa cells, oocytes). In humans, the expression pattern may be age and cycle dependent²⁵ and may have a role in ovarian embryogenesis.²⁶

TGF is also involved in the process of follicular apoptosis, which is central in maintaining a balance between cell proliferation and demise. Treatment of cultured granulosa²⁷ or theca-interstitial cells²⁸ with TGF inhibits the spontaneous onset of apoptotic DNA cleavage.

Transforming growth factor-β1 (TGF-β1), a homodimeric polypeptide comprising two identical 112-amino acid chains, is well recognized as a polyfunctional regulatory molecule.²⁹ Originally identified by its ability to elicit an acute reversible phenotypic transformation of normal mammalian cells, TGF-β1 has been shown to exert numerous regulatory actions in a variety of normal and neoplastic cells. At the level of the ovary, wherein it is produced,³⁰ TGF-β1 profoundly alters the proliferation and differentiation of rat granulosa cells.^31,32,33,34 An increasing body of evidence suggests that the ovarian theca-interstitial and granulosa cells may be sites of TGF-β1 production and action.³⁵ Further studies raise the possibility that TGF-β1 is involved in key events in ovarian life cycle from fetal and neonatal periods,³⁶ ovarian morphogenesis,³⁷ follicular maturation,³⁸ and rupture³⁹ and ultimately luteinization.⁴⁰

Basic fibroblast growth factor (bFGF), a 146-amino acid polypeptide, is a mitogen for a wide variety of mesoderm-derived and neuroectoderm-derived cells. Its complete isolation and characterization has been accomplished from various organs; an amino terminally truncated form lacking the first 15 residues was identified in the ovarian corpus luteum.⁴¹ Although the physiologic relevance of bFGF to ovarian function remains under investigation,⁴² several lines of evidence suggest that bFGF may play a central role in supporting the growth and development of the granulosa-luteal cell. Basic FGF constitutes the main mitogenic factor isolated from crude extract and has previously been shown to stimulate the replicative lifespan of cultured granulosa cells of bovine, porcine, rabbit, guinea pig, and human origin.^43,44,45 Because ovarian bFGF expression was not considered to be of granulosa cell origin,⁴⁶ whereas FSH induces functional receptors for bFGF in the granulosa cells,⁴⁷ it is tempting to speculate that locally produced bFGF⁴⁸ may play autocrine or paracrine regulatory roles at or adjacent to its sites of synthesis. In so doing, it may participate in the differentiation and replication of the developing granulosa cell.⁴⁹

Basic FGF is involved in early development of the human reproductive tract⁵⁰ and partakes in suppression spontaneous onset of apoptosis.⁵¹ The latter may be associated with the ability of progesterone to maintain granulosa cell viability.⁵² In addition to granulosa cells, bFGF also inhibits apoptosis of the ovarian surface epithelial cells, acting in both sites on its own receptor.⁵³ Basic FGF can be identified in a host of ovarian components, including granulosa cells, oocytes, follicular basement membrane, and surface epithelial cells.^54,55,56

Activin is a 24-kd protein with structural homology to TGF-β1. It was discovered during the purification of inhibin and found to be a dimer of the β subunits of the heterodimeric inhibin molecule.⁵⁷ Activin was concurrently discovered as capable of differentiating erythroleukemia cells⁵⁸ and inducing mesoderm formation.⁵⁹ Its presence in a variety of cell types suggests that it may regulate growth and differentiation in other tissues as well.⁵⁷

Activins play a role in the local regulation of ovarian function. Acting through a set of receptors, postulated to be membrane-bound serine/threonine kinases,^60,61 activin alters the function of granulosa and theca-interstitial cells. For instance, activin treatment of cultured granulosa cells from immature follicles increases FSH-supported estradiol production, inhibin production, and FSH and luteinizing hormone binding. Activin may maintain the immature follicle during the period of declining FSH levels, which is induced by its partner inhibin. In contrast to its action on immature granulosa cells, activin decreases progesterone production by mature granulosa cells from preovulatory follicles.⁶² Based on these observations, Findlay and coworkers⁶³ proposed an autocrine role for activin as a suppressor of spontaneous luteinization. Paracrine actions of activin are also a possibility because activin reduces luteinizing hormone-induced androstenedione production by cultured thecainterstitial cells.^64,65

Follistatin, a glycoprotein with isoforms of 35 to 40 kd, was also discovered during the purification of inhibin.⁵⁷ Its ability to bind activin⁶⁶ provides a possible explanation for the observation that follistatin antagonizes the in vitro actions of activin. The presence of follistatin primarily in preovulatory follicles⁶⁷ supports the idea that blocking activin is necessary for maturation and luteinization. In the developing follicles, FSH-induced granulosa cell proliferation and mitogenesis is facilitated by activin.⁶⁸

The study of activin action is further complicated by the ability of its component subunits to combine with the α subunit of inhibin to form a molecule whose action in many experimental assays is diametrically opposed to that of activin. Although autocrine actions of inhibin have not been convincingly demonstrated, granulosa cell-derived inhibin can oppose the activin blockade of thecal androgen production.⁶⁴ Activin, follistatin, and inhibin form a complex mix of intraovarian regulators (Fig. 5).

TNF-α, a 157-amino acid polypeptide, was originally named for its oncolytic activity as displayed in the serum of bacillus Calmette-Guérin-immunized, endotoxin-challenged mice.^92,93 TNF proved capable of inducing tumor necrosis in vivo and of exerting nonspecies specific cytolytic or cytostatic effects on a broad range of transformed cell lines in vitro. Although TNF was initially thought to be tumor selective, it has become clear that certain nontumor cells possess TNF receptors and that TNF may be a regulatory monokine with pleiotropic noncytotoxic activities in addition to its antitumor properties. TNF engages in the differentiation of a variety of cell types.

At the level of the ovary, TNF was found capable of attenuating the differentiation of cultured granulosa cells from immature rats.⁹⁴ In other studies, TNF was found to effect complex dose-dependent alterations in the elaboration of progesterone and androstenedione, but not estrogen, by explanted preovulatory follicles of murine origin. Although the ovary contains TNF mRNA,⁹⁵ its in vivo origins must be determined. In principle, two general possibilities are worthy of consideration. TNF may be locally derived from (activated) resident ovarian macrophages,⁹⁶ as shown for regressing (but not young) corpora lutea. Although basal TNF activity was undetected in corpora lutea of pregnancy and pseudopregnancy, TNF activity was markedly stimulated in the presence of lipopolysaccharide.⁹⁷ However, the detection of TNF activity in some luteal tissue on day 5, and the scarcity of macrophages at this stage raise the possibility that cells other than macrophages may also produce TNF in the corpus luteum. TNF may be of granulosa cell origin, as suggested by immunohistochemical studies wherein antral or atretic granulosa cells have been implicated as a possible site of TNF gene expression. Given such strong association between TNF elaboration and follicular and luteal decline, it is tempting to speculate that TNF may play a role in the still enigmatic processes of atresia or luteolysis. In this capacity, TNF of intraovarian origin may exert its effects at or adjacent to its site of synthesis, interacting with specific granulosa-luteal cell surface receptors to modulate gonadotropin hormonal action. TNF-induced luteolysis⁹⁸ by apoptosis has been well documented.⁹⁹ TNFinduced apoptosis depends on the ceramide signaling pathway as its second messenger.^100,101 Undoubtedly, future studies of the regulation of the TNF receptor and the elucidation of the in vivo source of its ligand will shed new light on the relevance of this system to the process of follicular development or demise.

Other growth and peptidergic factors have potential physiologic relevance to folliculogenesis (Table 1). The ovary contains a complete renin-angiotensin system that may be involved with vascularization and with modulation of steroidogenesis.¹⁰² Vasoactive intestinal peptide is also produced locally in the ovary, and it can enhance estrogen production by granulosa cells of prepubertal rats.¹⁰³

Nerve growth factor is another peptidergic factor whose mRNA has been detected in the ovary,¹⁰⁴ but its modulatory role in the ovary is unknown. Similarly, endothelin, a potent vasoconstrictor, influences ovarian progesterone production.¹⁰⁵

If there are any lessons to be learned at this time, it is that optimal gonadotropin hormonal action is highly contingent on the input of tissue-based regulatory principles. According to this view, gonadotropins may not be the omnipotent agents they were once thought to be. Rather, gonadotropins may best be viewed as “team players” and as initiators of a cascade of events facilitated, attenuated, or mediated through interaction with putative intraovarian regulators. It is the special case of IGF-I that best illustrates the role of a tissue-based modulator in that optimal gonadotropin hormonal action is clearly highly dependent on the availability of IGF-I and the consequent amplification of gonadotropin hormonal action.¹⁴ In contrast, putative intraovarian regulators exemplified by TGF-α may attenuate gonadotropin hormonal differentiation in the interest of continued proliferative ability.

Another role for putative intraovarian regulators, exemplified by IL-1, is that of mediation of gonadotropin action. According to this view, IL-1 constitutes an extension of the gonadotropin signal, possibly one of several more distal effectors, the overall mission of which may well be the conveyance of the message (or portions thereof) imparted by the midcycle surge.

The development of the ovarian follicle is a continuum of growth and differentiation of at least three distinct cell types: thecal cells, granulosa cells, and oocytes. Much depends on the localization and timing of expression of the regulatory principles. Of equal importance is the ability of the target cell to receive and respond to the regulatory signal. Activin elicits a stimulatory and an inhibitory response, depending on the cell type being studied⁶⁴ and the developmental stage of the follicle.⁶² This duality of action may be explained if the activin receptor isoforms⁶¹ prove to have a specific cell-type and developmental-stage distribution. The action of a given regulatory factor, such as EGF/TGF, can also be influenced by the presence or absence of other factors.¹⁰⁶ The ability of IL-1¹⁰⁷ and nerve growth factor¹⁰⁸ to alter EGF/TGF binding in nonovarian cell types is an example of how one growth factor can impinge on the actions of another.

It is the net balance representing the integration of multiple transduction pathways (Fig. 7) and often opposing signals that determines final gonadotropin hormonal action. Moreover, a given intraovarian growth factor may play several roles, depending on its local concentration, availability of its receptors or binding proteins, the cell population with which it interacts, and the precise timing of that interaction. There is every reason to believe that future studies may reveal other modes of interaction between trophic ovarian principles and tissue-based regulatory elements. It is with a strong sense of excitement that future work in this evolving area is anticipated.

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